Millions of women are diagnosed with urinary tract infections each year. Countless numbers of dogs and cats also suffer from chronic urinary infections and die from renal infections. E. coli is the most common pathogen associated with these infections, causing over 80% of urinary tract infections. Over 30% of women suffer recurrent infections within a 6 to 12-month period and are forced to resort to extended use of antibiotics to treat these infections. Recurrent use of antibiotics can lead to pathogen resistance and result in deleterious side effects and toxicity reactions. Consequently there exists a need for safe alternative medications (e.g., non-antibiotics) that can be used to prevent or treat urinary tract infections in both animals and humans. Moreover, urinary tract inflammation is a painful and often debilitating symptom of bacterial infection as well as many diseases of both known and unknown etiology. Thus, there is currently a need for new treatments to effectively mitigate the pain arising from inflammation of the urinary tract.
The two main forms of urinary tract infection are the renal infection known as pyelonephritis and the bladder infection referred to as bacterial cystitis. As the bladder is closer to the anus, the site of entry for bacteria giving rise to urinary tract infections, cystitis is far more common than pyelonephritis. Moreover, although cystitis is not as deadly as its renal counterpart, it is associated with pervasive inflammation giving rise to severe bladder pain as well as frequent, urgent, and painful urination. Cystitis is also often a recurrent condition, resulting in long term and often debilitating discomfort for those afflicted.
Unfortunately, the causes of the battery of adverse conditions affecting the urinary tract are only partially understood. Thus, inflammatory conditions of the bladder of both known and unknown etiology are commonly referred to as cystitis. As noted above, cystitis may be bacterial in nature, arising from infection of the bladder by E. coli, Cystitis may also embody urinary tract inflammation due to a number of non-bacterial sources, for example, allergic responses to food or insufficient water intake, which allows the bladder and urethral tissues to become dry, thus leading to deposition of crystallized uric acid on the tissues and associated irritation. Interstitial cystitis is a recurrent condition of uncertain etiology. It is a source of frustration for both doctors and patients alike, as it has no apparent infective cause and is frequently debilitating. Indeed, according to the first epidemiological study of interstitial cystitis in the U.S. (Held, et al. 1990), 50% of patients could not maintain full-time employment due to the painful effects of the disease.
Treatments for cystitis include antimicrobials, anti-inflammatory agents, buffering agents, muscle relaxants, mast cell stabilizers, painkillers including tricyclic antidepressants and transcutaneous electrical nerve stimulator (TENS) units, catheterization and intravesicular instillation of heparin, the anti-inflammatory agent dimethyl sulfoxide, the detergent sodium oxychlorosene, the caustic agent silver nitrate, or chromolyn sodium, and surgical techniques. Unfortunately, many of these treatments are themselves painful, and many are also unsuitable for personal treatment in the home.
The fruit of the American cranberry (Vaccinium macrocarpon) has received considerable attention for its putative human health benefits. Most of the focus is on the flavonoid constituents due to their relatively high biological activity in various assays. In vitro chemical assays have rated cranberries as having some of the highest antioxidant values of over 21 fruits (Vinson et al. 2001; Sun et al. 2002), and the overall phenolic content appears to correlate with the level of antioxidant activity. The ‘Folin-Ciocalteu’ colorimetric test has found cranberry to have one of the highest phenolic contents of a number of fruit species tested (Vinson et al. 2001; Sun et al. 2002). The phenolic classes identified in cranberry include phenolic acids (Marwan et al. 1982; Heimhuber et al. 1990; Zheng et al. 2000; Zuo et al. 2002), anthocyanins (Hong et al. 1986; Hong et al. 1990), flavonols (Puski et al. 1967; Yan et al. 2002), and flavan-3-ols, which consist of both monomers and the polymer classes procyanidins and proanthocyanidins (Foo et al. 1981; Foo et al. 2000a; Foo et al. 2000b; Cunningham et al. 2002). As described more fully below, we have previously identified cranberry A-type proanthocyanidins as possessing anti-adherence activities against uropathogenic type P E. coli (Foo et al. 2000a; Foo et al. 2000b; U.S. Pat. No. 6,608,102).
Cranberry juice has been shown to reduce bacteriuria associated with urinary tract infections in humans (Avorn et al. 1994). One mechanism implicated in yielding this beneficial effect is the ability of certain compounds present in cranberries to inhibit the adhesion of type 1 and type P fimbriated E. coli to human epithelial cells (Sobota 1984; Schmidt et al. 1988; Zafriri et al. 1989). Type P fimbriated E. coli have been implicated as the main cause of pyelonephritis, while type 1 fimbriated E. coli are the predominant causative agent of bacterial cystitis.
Zafriri et al. (1989) reported that fructose present in cranberry juice inhibited the adherence of type 1 E. coli to uroepithelial cells, and that cranberry juice also contained one or more non-dialyzable substances that inhibited binding of type P E. coli but failed to define the chemical nature of those substances.
U.S. Pat. Nos. 5,474,774, 5,525,341, and 5,646,178 to Walker et al. disclose cranberry extracts having the ability to inhibit the adherence of E. coli to uroepithelial cells. This activity was obtained by extracting whole cranberries with acidified alcohol followed by separation of the activity from simple sugars by precipitation with a metal acetate or sulfate. Upon further manipulation, the reported activity consisted of a fraction enriched in polyphenol and flavonoid compounds that contained as much as 10% anthocyanins. The specificity of this anti-adherence activity for type 1 or type P E. coli was, however, not determined.
WO 96/30033 and U.S. Pat. Nos. 5,646,178 and 5,650,432 to Walker et al. disclose a series of proanthocyanidin monomers, dimers, and polymers as well as flavonoid derivatives thereof and related compounds purported to have the ability to interfere with bacterial adherence to a surface. The dimers and polymers of Walker were limited to compounds having B-type interflavanoid linkages. However, Walker failed to provide any experimental data correlating biological activity with a specifically identified compound. The extraction method involved alkalinizing a plant material homogenate to a pH greater than 10 (a treatment which causes degradation of proanthocyanidins) and precipitating the polyphenolic compounds (together with other materials) by addition of alcohol. This precipitate contained the proposed anti-adherence activity and was further fractionated to yield the purified active compound. Using this process with an aqueous solution of commercially available Ocean Spray cranberry powder, Walker reported obtaining a single active compound and partially characterized the compound but failed to provide its chemical structure. The Walker assay methods could not distinguish between anti-adherence activities with respect to type 1 or type P E. coli, thus Walker was also unable to characterize the biological activity of this compound.
Commonly assigned U.S. Pat. No. 6,608,102 to Howell et al. discloses plant proanthocyanidin extracts that are substantially free of anthocyanins and flavonols and specifically inhibit the adherence of type P E. coli to epithelial cells. The Howell '102 patent represents the culmination of our work that conclusively demonstrated that proanthocyanidins are the chemical agents in cranberries and other plants that are responsible for this anti-adherence activity, and that proanthocyanidins having at least one A-type interflavanoid linkage are particularly potent agents for this activity. Later U.S. Pat. Nos. 6,210,681 and 6,440,471 to Walker et al. disclose similar proanthocyanidin extracts.
Thus, we have previously determined that proanthocyanidins present in cranberries exhibit anti-adherence activity with respect to the binding of uroepithelial cells solely by type P fimbriated E. coli, However, as bladder infections are associated with type 1 fimbriated E. coli, the anti-adherence activity of these compounds is ineffective for the treatment of bacterial cystitis. Although fructose in cranberry juice has been found to possess an analogous anti-adherence activity with respect to the binding of type 1 fimbriated E. coli to uroepithelial cells and is therefore theoretically effective at hindering bladder infections, this discovery was made well over a decade ago, yet there remains a substantial need for efficacious treatments for cystitis.
Cranberries and cranberry products have been used in the treatment of urinary tract infections both alone and in conjunction with other therapies. The basis for such treatments has traditionally implicated the antimicrobial properties associated with cranberries. Originally, this antimicrobial activity was thought to arise from acidification of the urine due to the intake of cranberry juice, resulting in an unfavorable environment for bacterial survival. Later, the focus shifted to the bacterial anti-adherence activity of fructose and proanthocyanidins that has been elucidated in the aforementioned literature. For example, Cystopurin, a Roche product for the treatment of cystitis that is available in the UK, incorporates a cranberry juice extract as an adjunctive therapy in a potassium citrate buffer designed to alleviate painful urination by neutralizing urinary acidity. However, as noted above, the only constituent of cranberries known to possess anti-adherence activity against the type 1 fimbriated E. coli commonly involved in bacterial cystitis is fructose. Moreover, inflammation of the urinary tract may also arise from non-bacterial sources, in which case such anti-adherence activities are therapeutically ineffective. Finally, individuals having non-bacterial cystitis must exercise care in the process of self-medication with cranberry products, as, depending on the nature of the preparation, acids and other bladder irritants may be present that could result in aggravation of the inflammatory condition.
After bacterial adherence, internalization of type 1 E. coli by bladder epithelial cells represents another potential target for the treatment of bacterial cystitis. Traditional thinking had considered uropathogenic E. coli to exist as extracellular pathogens within the urinary tract, despite the fact that transmission electron microscopy studies of infected rat and mouse bladders indicated that bladder epithelial cells could internalize the pathogens in vivo (Fukushi et al. 1979; Mc Taggart et al. 1990). Although such internalization was initially regarded as a host defense mechanism, Mulvey et al. (1998) suggested that it inured beneficially to the survival of the bacteria. Specifically, they determined that type 1 E. coli induced programmed cell death and exfoliation of bladder epithelial cells, however, pathogens could avoid expulsion by such mechanisms by invading into deeper tissue. They also speculated that the frequency of recurrence of infection despite antibiotic treatment could be linked to the persistence of bacteria within the cells of the bladder long after the death of extracellular pathogens by such treatments. This speculation was later confirmed by their discovery that a persistent reservoir of E. coli could be established by bacterial invasion of bladder epithelial cells, followed by intracellular replication and reemergence of the pathogens (Mulvey et al. 2001). Upon reemergence, the pathogens evade clearance via exfoliation of the infected bladder cells by anchoring themselves and invading into deeper, healthy cells that become exposed to the bladder lumen as a result of the exfoliation of cells from more superficial layers. These internalized pathogens can persist in a latent state and, as the result of an as yet undetermined trigger, reemerge to cause recurrent infections.
Martinez et al. (2000) reported that the FimH element, an adhesin located on the tip of the type 1 pilus, mediates the bacterial invasion of human bladder epithelial cells. Specifically, they determined that the FimH element induces rearrangements of the host cell cytoskeleton resulting in a zippering effect by which the host cell engulfs the pathogen. Martinez also reported that these cytoskeletal rearrangements required protein tyrosine phosphorylation and phosphoinositide 3-kinase activation, and that a quercetin derivative, LY294002 (Vlahos et al. 1994), was a potent inhibitor of both of these activities and effectively inhibited type 1 pilus-mediated bacterial invasion of bladder epithelial cells in urinary tract infections. Tyrosine kinase inhibitors, including both quercetin and myricetin, directly inhibit enzymes such as the hexose transporter GLUT1 via specific competition for the ATP binding site (Vera et al. 2001). Myricetin has been found to be the most potent flavonol for inhibiting transport of methylglucose and deoxyglucose, while iso-rhamnetin (3′-methoxyquercetin) was the most potent at inhibiting transport of dehydroascorbic acid (Vera et al. 2001).
Quercetin has attracted much attention for its potential health benefits, and has been associated with numerous biological activities, including anti-inflammatory activities (Formica et al. 1995). Quercetin and related compounds inhibit a number of the processes associated with inflammation including lipopolysaccharide induced production of nitric oxide and tumor necrosis factor a (TNF-α) (Kawada et al. 1998; Wadsworth et al. 1999) and cytokine production (Xagorari et al. 2001). The invasion of bladder cells by type 1 E. coli has been shown to induce cytokine production by a lipopolysaccharide dependent mechanism (Schilling et al. 2001). Among fruit species, the cranberry contains one of the highest concentrations of quercetin, ranging from 11 to 25 mg/100 g of fresh fruit (Bilyk et al. 1986; Hakkinen et al. 1999). Quercetin is predominantly found in a conjugated form with various sugars, and the sugar moiety may significantly influence its bioavailability and adsorption (Hollman et al. 1995; Hollman et al. 1999; Woffram et al. 2002).
Quercetin has been used in the treatment of inflammatory conditions associated with the urinary tract. U.S. patent application Ser. Nos. 757358 and 848187 to Katske, et al. disclose compositions and methods for the treatment of non-bacterial prostatitis and non-bacterial cystitis, respectively. These compositions comprise bioflavonoids having a substantial percentage of quercetin that exhibit both anti-oxidative and anti-inflammatory properties and a digestive enzyme such as bromelin or papain to increase the intestinal absorption of the bioflavonoid component. The compositions are directed towards treating the pain associated with cystitis and prostatitis by an anti-inflammatory mechanism. However, substances such as papain are allergenic to many individuals, and these enzymes have caustic and corrosive effects on the digestive mucous membranes.
Thus, there is presently a need for non-antibiotic treatments for urinary tract infections. There is also a need for a generalized therapy for cystitis, whether bacterial or non-bacterial in origin, that is innocuous and can be used for self-medication in the home. Accordingly, applicants have discovered that the cranberry flavonol compositions of the present invention advantageously possess superior anti-inflammatory activity. These compositions are amenable to use in the treatment of inflammatory disorders, particularly those of the urinary tract arising from both bacterial and non-bacterial sources. Moreover, these compositions can be formulated to contain one or more flavonols that can inhibit the invasion of uroepithelial cells by type 1 fimbriated E. coli, Advantageously, these compositions are also free from irritants of the bladder as well as the digestive mucous membranes. Thus, the cranberry flavonol compositions of the present invention embody natural, innocuous treatments that can be used beneficially for self-medication by those afflicted with inflammatory conditions of the urinary tract generally, and cystitis particularly. The cranberry flavonol compositions of the present invention are effective for and amenable to the treatment of such inflammatory conditions regardless of whether such conditions originate from bacterial or non-bacterial sources.